The autophagy machinery is
required to initiate hepatitis C
virus infection
Marlène Dreux
Hepatitis C virus life cycle
HCV particles
internalization
RNA
replication
RNA
translation
HCV assembly
and release
ER
Adapted from Tibotec
2
Hepatitis C virus life cycle
?
ER
Transport and delivery of the incoming HCV genome to the requisite
cellular factors and location to initiate translation and replication.
3
Autophagy
•  Evolutionarily conserved pathway that maintains cellular homeostasis
•  Evolutionarily conserved pathway that maintains cellular homeostasis
Cytoplasm
Autophagosome
expansion
ER
Nucleus
4
Autophagy
Endosomal compartment
•  Evolutionarily conserved pathway that maintains cellular homeostasis
Cytoplasm
Autophagosome
expansion
ER
Nucleus
Autolysosome
degradation
5
Autophagy and viral infection
• • Elimination
of intracellular pathogens, xenophagy
Evolutionarily conserved pathway that maintains cellular homeostasis
e.g. HSV, Sindbis virus
•  Regulation of host response to virus infection
e.g. modulation of RIG-I- and TLR- induced signaling, antigen presentation
Autophagy is emerging as a central component of
antimicrobial host defense
•  Pathogens evade, subvert, or exploit autophagy
e.g. γ-Herpesvirus, poliovirus, HIV, Influenza virus
Autolysosome
degradation
Orvedahl et al. Cell Host & Microbe 2010, Lee et al. Science 2007, Shelly et al. Immunity
2009, Talloczy et al. Autophagy 2006,
ER
Tal et al. PNAS 2009, Jounai et al. PNAS 2007, Orvedahl et al. Cell Host & Microbe 2007, Jackson et al. Plos Biol 2005, Kyei et
al. J Cell Biol 2010,
Gannage et al. Cell Host & Microbes 2010.
Nucleus
6
Autophagy and viral infection
• • Elimination
of intracellular pathogens, xenophagy
Evolutionarily conserved pathway that maintains cellular homeostasis
e.g. HSV, Sindbis virus
•  Regulation of host response to virus infection
e.g. modulation of RIG-I- and TLR- induced signaling, antigen presentation
Autophagy is emerging as a central component of
antimicrobial host defense
Possible role of autophagy in
the HCV life cycle?
ER
Nucleus
Autolysosome
degradation
7
Does HCV regulate autophagy?
•  Evolutionarily conserved pathway that maintains cellular homeostasis
LC3
LC3-II
LC3-II
Autophagosome
expansion
ER
Nucleus
Autolysosome
degradation
8
Enhanced autophagic vesicle content
in HCV-infected cells
mock
HCV
Autophagy
inducers
β-actin
HCV
Tg
ALLN
b
LC3
LC3-II
% cells with
punctate LC3-GFP:
77%
28%
•  LC3 redistribution into discrete
dots increases in infected cells
•  HCV increases the conversion
of LC3 to LC3-II
Ait-Goughoulte et al. J Virol, Ferraris, et al. JGV, Ke and Chen JCI, Sir et
al. Hepatology, Rautou et al. Am. J. Pathol. 2011
9
Does autophagy perform an antiviral or proviral
function during HCV infection ?
•  Evolutionarily conserved pathway that maintains cellular homeostasis
Vps34
Beclin1
Atg4B
Atg12
Atg4B
Autophagosome
expansion
ER
Autolysosome
degradation
10
Does autophagy perform an antiviral or proviral
function during HCV infection ?
Beclin-1
Atg4B
control sh49 sh50
control sh800 sh801
Atg4B
•  Evolutionarily conserved pathway that maintains
cellular homeostasis
Beclin-1
β-actin
β-actin
Vps34
Beclin1
Atg12 shRNA
Tg
Atg4B
-  +
-  -
+
+
+
LC3
LC3-II
Atg12
Atg4
Autophagosome
expansion
ER
Autolysosome
degradation
11
Autophagy regulators are required
for the HCV life cycle
•  Infection of autophagy protein deficient cells
extracellular infectivity (ffu/ml)
•  Analysis of viral expansion
control
-
1.E+5
shRNA-49
shRNA-50
Beclin-1
-control
shRNA-800
shRNA-801
Atg4B
Atg12
-control
shRNA-92
1.E+4
1.E+3
1.E+2
1.E+1
1
2
3
4
5
time (days)
6
1
2
3
4
5
time (days)
6
1
2
3
4
5
6
time (days)
12
Identification of the autophagy-dependent
step in the HCV life cycle.
Cell entry
RNA replication
Translation
Virus assembly
13
Accumulation of intracellular HCV RNA depends on
autophagy proteins
•  Single cycle infection (MOI of 10, 24 hours)
Infectivity
100
50
0
shRNA:
-
GFP
Beclin-1 Atg4B
Atg12
HCV RNA
Atg4B
control
Atg12
100
Intracellular RNA
intracellular infectivity
extracellular infectivity
50
0
shRNA:
-
GFP Beclin-1 Atg4B Agt12
14
Accumulation of intracellular HCV RNA depends on
autophagy proteins
•  Single cycle infection (MOI of 10, 24 hours)
HCV RNA
Atg4B
control
Atg12
Intracellular
RNA RNA
Intracellular RNA
100
100
HCV RNA
50
50
0
shRNA:
0
shRNA:
-
GFP Beclin-1 Atg4B Agt12
control Beclin-1
Agt12
Atg4B
Autophagy machinery proteins regulate either virus
entry, RNA translation or replication
15
HCV cell entry does not depend on
autophagy proteins
HCV pseudoparticles
HCV E1/E2
Entry analysis
shRNA:
Autophagy proteins are required at a post entry
step, most likely HCV RNA translation or replication.
16
HCV translation/replication
luciferase
Autophagy machinery proteins are required to
initiate RNA translation or replication
17
Translation of incoming HCV RNA
replication-defective construct
(inactivation mutation in the active
site of viral polymerase)
-Rluc
shRNA: control
Autophagy machinery is required for translation of
incoming HCV RNA
18
Are autophagy proteins required to maintain HCV translation and
replication in persistently infected cells ?
HCV
infection
Virtually all infected cells
shRNA vectors
8 days
Analysis
(protein expression,
infectivity
and HCV RNA level)
shRNA:
19
Are autophagy proteins required to maintain HCV translation and
replication in persistently infected cells ?
HCV
infection
Virtually all infected cells
shRNA vectors
8 days
Beclin-1
Atg12
- sh
- sh49 sh50
shRNA:
Beclin-1
β-actin
Atg4B
- sh800 sh801
Analysis
(protein expression,
infectivity
and HCV RNA level)
β-actin
LC3
LC3-II
Atg4B
β-actin
Autophagy machinery is required for translation of incoming RNA
and, therefore, HCV replication in de novo infected cells, but not for
translation of progeny HCV RNA once replication is established.
20
Conclusion
• Key autophagy regulators are required for productive HCV infection
• Different host factors regulate the translation of incoming viral
genome vs. the translation of progeny HCV RNA once replication is
established.
• These results have fundamental importance because they provide
mechanistic insights into a hitherto unstudied step of HCV
infection.
Autophagy proteins might contribute to the transport/delivery of HCV
genome towards the translation apparatus.
Alternatively, since HCV RNA translation necessary takes place in the
ER, ER sub-domains identified as a source of membranes for formation
of autophagy vesicles could be used as membrane support for
21
translation of incoming HCV RNA.
Many thanks to...
National Institute of Infectious Diseases,
Tokyo
Takaji Wakita
Rockefeller University, New York
Charles M. Rice
The Scripps Research Institute
Francis V. Chisari
Pablo Gastaminza
Stefan Wieland
The Scripps Research Institute
Dennis Burton, Mansun Law
Salk Institute, La Jolla, CA
Inder Verma
Ecole Normale Superieure, Lyon
François-Loïc Cosset
Present address
Virology Department, Ecole Normale supérieure, INSERM U758, Lyon, France